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Journal of Clinical Microbiology, February 2002, p. 501-507, Vol. 40, No. 2
0095-1137/01/$04.00+0     DOI: 10.1128/JCM.40.2.501-507.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Rapid Detection of Pyrazinamide-Resistant Mycobacterium tuberculosis by a PCR-Based In Vitro System

Yasuhiko Suzuki,^1* Aya Suzuki,¹ Aki Tamaru,² Chihiro Katsukawa,² and Hajime Oda³

Departments of Pathology,¹ Microbiology,² Industrial Health, Osaka Prefectural Institute of Public Health, Higashinari-ku, Osaka 537-0025, Japan³

Received 9 July 2001/ Returned for modification 30 August 2001/ Accepted 28 October 2001

Pyrazinamide (PZA), an analog of nicotinamide, is a prodrug for tuberculosis which requires conversion to the bactericidal compound pyrazinoic acid by bacterial pyrazinamidase activity. Mutations leading to a loss of pyrazinamidase activity cause PZA resistance in Mycobacterium tuberculosis. Thus, the detection of pyrazinamidase activity makes the discrimination of PZA-resistant tuberculosis possible. However, the detection of the pyrazinamidase activity of M. tuberculosis isolates needs a large amount of bacilli and is therefore time consuming. In this paper, we describe a new method for the detection of pyrazinamidase activity with a PCR-based system. The genes encoding pyrazinamidase (pncA genes) in 30 resistant clinical isolates were amplified by PCR by using forward primers containing bacteriophage T7 promoter sequences at their 5" ends. Then the PCR products were directly subjected to an in vitro transcription-translation coupled system. All of the PZA-resistant isolates tested showed reduced pyrazinamidase activity compared to susceptible M. tuberculosis type strain H37Rv. In contrast, all of the 15 susceptible clinical isolates exhibited pyrazinamidase activities similar to that of H37Rv. This fact suggested the possibility of the usefulness of this system for the rapid detection of PZA-resistant M. tuberculosis.

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* Corresponding author. Mailing address: Department of Pathology, Osaka Prefectural Institute of Public Health, Nakamichi 1-3-69, Higashinari-ku, Osaka 537-0025, Japan. Phone: 81-6-6972-1321, ext. 268. Fax: 81-6-6972-0772. E-mail: suzuki{at}iph.pref.osaka.jp.

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Journal of Clinical Microbiology, February 2002, p. 501-507, Vol. 40, No. 2
0095-1137/01/$04.00+0     DOI: 10.1128/JCM.40.2.501-507.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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